Académique Documents
Professionnel Documents
Culture Documents
Hugo Osrio
hosorio@ipatimup.pt
PROTEOMICS
Proteomics: Prote - Protein
Omics - Total
Protein identification
Characterization: Post-translational modifications PTMs
Cells
2011 5428
2001 522
1998 22
1997 1!
Organs
Organisms
Tissues
Cancer
Medical research
Neurosciences
Psychiatry
Cardiology
Biological research
Yeast
Plants
Fish
2D gel electrophoresis
Separation of Human Serum proteins by 2D gel electrophoresis
Isoelectric point
3
MW (kDa)
250
150
100
75
50
37
25
10
www.nonlinear.com/products/progenesis/samespots/overview/
www.nonlinear.com/products/progenesis/samespots/downloads/
Alignment
Results
Data analysis
www.biorad.com/prd/en/US/LSR/PDP/966de
b78-2656-437f-b7a4ab0a9bd45c8d/PDQuest-2-DAnalysis-Software
www.gelifesciences.com/webapp/
wcs/stores/servlet/productById/en/
GELifeSciences-PT/28940830
Mass spectrometry
Ionization Source
Mass analyzer
Generates ions
Separates ions
Detector
Detects ions and
amplifies the signal
Sample + Matrix
+
+
MALDI
Matrix Assisted Laser Desorption Ionization
+
+
-Cyano-4-hydroxycinnamic acid
Sinapinic acid
2,5-dihydroxybenzoic acid
MALDI
Laser
mass/charge
Detector
16953.3 Da
100
1 2 5 1 .1
90
80
70
% In te n s ity
60
50
40
30
Thioredoxin
11674.5 Da
20
10
0
9 9 8 5 .0
1 1 6 3 1 .4
1 3 2 7 7 .8
1 4 9 2 4 .2
M a s s (m /z )
1 6 5 7 0 .6
1 8 2 1 7 .0
Plate: 1 Spot: F2
120000
1821.91
1821 Da
110000
100000
1688 Da
90000
1688.86
948 Da
948.45
80000
1781.86
C l u s te r A r e a
70000
1733.86
60000
50000
2132.06
1659.84
40000
1958.06
30000
1388.77
20000
3836.78
764.43
893.45
10000
1025.51
1073.52
1611.85
823.48
1641.76
842.51
871.48
1028.94
1089.51
970.44 1116.52
1578.83
1402.78
1825.90
2211.11
1888.93
1919.96
1488.70
3850.84
2445.01
2225.12
2807.32
2189.09
1878.93
3014.48
0
1000
1500
2000
2500
Mass
3000
3500
100
5281.6
5673.1226
5761.1216
90
5409.1899
5365.4541
5805.1416
5892.9102
80
5849.4219
5321.2124
5277.4556
70
5937.0415
5980.6997
6024.8804
% Intensity
60
5189.7905
5145.6392
6068.6787
6113.2207
50
6157.3066
5101.9717
40
6245.3008
5014.1318
4970.2163
6288.8379
30
20
10
0
2991
4018
5045
6072
M ass (m/z )
7099
8126
Protein identification
2D Gel
+
pI
MW
Membrane transfer
Western Blot
Peptide extraction
100
7.3E+4
90
80
929.370
70
60
50
40
930.371
30
944.362
20
910.366
Protein
10
0
860
931.371
945.368
912.361
932.368
946.371
960.359
898.471
882.529
938.756
914.369
920.867
868.501
927.758
953.767 967.531
905.508
875.680
891.042
892
924
956
Mass (m/z)
Bioinformatic analysis
1050 Da
900 Da
996.546
982.551
989.823
988
1009.215
1020
1298.651( R 11281,S906)
1.5E+4
90
80
70
60
50
1088.519( R 9327,S508)
1210.607( R 10791,S460)
1901.838( R 17349,S332)
880.417( R 8953,S490)
40
1906.829( R 16997,S261)
898.442( R 7544,S370)
MS - INTERNAL CALIBRATION
340000
1994.885( R 18017,S233)
2126.984( R 17889,S218)
30
Statistical analysis
Plate: 1 Spot: F9
320000
1213.64
300000
1377.615( R 11118,S185)
280000
1835.96
260000
1771.929( R 16092,S138)
20
240000
1136.615( R 9246,S155)
220000
1361.8
2024.6
2687.4
3350.2
Plate: 1 Spot: E3
200000
C l u s te r A r e a
220000
180000
MS - INTERNAL CALIBRATION
Plate: 1 Spot: H1
160000
200000
1298.65
140000
200000
842.51
180000
896.47
120000
4013.0
100000
160000
Mass (m/z )
80000
180000
1540.73
1287.63
60000
140000
2211.10
40000
842.51
1153.61
1243.65
20000
926.48
1082.58 1181.61
788.41
C l u s te r A r e a
719.35
818.41
1034.54
3168.50
1882.02
940.49
120000
1117.63
1375.62
1475.77
2225.12
1818.93
1592.85
1338.70 1451.74
1307.69
1666.82
1269.68 1389.65 1564.79
1624.87
1791.75
1896.03
1761.87 1935.98
1983.02
2383.95 2490.19
2593.12
2337.14
2457.16
2275.01
2316.13
160000
2705.15
3223.32
0
1000
1500
2000
100000
MS - INTERNAL CALIBRATION
2500
3000
3500
Mass
Plate: 1 Spot: H5
160000
80000
150000
140000
842.51
140000
870.54
60000
927.49
1062.55
120000
120000
40000
1479.77
745.33
1011.49
1286.65
110000
1639.90
830.46
1880.90
1234.66
MS - INTERNAL CALIBRATION
1274.60
896.47
20000
1003.56
763.39
1320.57
1161.59
1995.93
1045.55
797.42
100000
1088.52
962.49
C l u s te r A r e a
1920.91
1592.81
1521.75
1439.74
90000
Plate: 1 Spot: H3
1567.72
1419.65
1390.66
1126.50
927.49
1439.75
1213.61
1193.59 1357.68
1062.52
280000
1538.77
2211.11
1707.75
2225.12
1858.87
2343.21
2096.10 2125.94
2496.38
1677.77 1793.96 1982.93
2282.09
1954.02 2062.92
2383.97
1747.79 1716.80 1816.90
2161.00 2258.01
2033.92
2420.09
2612.21
2545.17
2572.37
2801.34
2748.40
2718.17
1088.52
3083.49
3246.62
1900.84
1210.61
100000
2874.46
3408.67
1639.93
260000
C l u s te r A r e a
1904.93
130000
1000
1500
2000
2500
3000
3500
1427.68
4000
Mass
80000
828.33
240000
70000
870.55
220000
80000
60000
880.42
927.50
200000
1993.88
50000
2062.94
1612.89
40000
180000
C l u s te r A r e a
60000
1319.68
1287.67
30000
160000
1003.59
2125.93
719.37
842.51
805.40
2328.07
1904.95
1083.58
1770.93
1479.80
1516.64
960.56
1298.65
875.51
982.46
20000
1149.59
140000
1563.86
1915.95
1193.59
1140.59
1029.57
120000
1251.66
1211.59
1213.63
MS - INTERNAL CALIBRATION
1677.80
1355.69
1399.70
1660.71
1793.88
1719.87
1951.96
2062.94
2495.26
2196.11
2395.13
2046.00
1835.93
2153.94
1735.83 1771.92
1000
1003.59
2125.98
1612.78
2874.38
2011.93
1428.69
100000
1253.62
1523.81
1114.63
898.45
2096.10
1880.93
759.32
10000
1374.50
2211.11
1639.94
1088.52
2282.05
2511.02
2612.19
40000
3194.33
2541.28
2574.38
2786.68
2749.36
3045.47
3350.43
3095.34
1136.61
3280.43
1505.72
1500
2000
2500
896.47
3000
842.51
2496.34
3500
Mass
1456.68
65000
1287.66
80000
1179.60
830.45
20000
1612.78
1475.75
1062.54
60000
1029.58
1904.94
1791.73
1511.83
1320.60 1427.67
719.37
759.44
1835.95
1707.78
785.34
1677.79
2383.97
2496.34
50000
1377.61
20000
1552.67
1880.92
1819.89
1740.74
1561.75
1914.93
1657.81
45000
736.39
1393.60
2782.32
2211.11
2329.08
2705.19
2225.12
2289.99
2560.09
2082.99
2689.16
2399.05
2874.41
1952.96
2248.03 2343.00
2749.32
2096.08
2541.25
2817.36
2573.36
796.36
842.49
1475.76
1791.76
1657.80
1628.77
1522.72
1552.67
1707.78
1852.84
1975.93
1682.97
1861.84
3480.67
1000
3619.92
1500
2000
2500
3000
2084.00
2034.97
1734.80
1500
2290.00
2383.97
2212.12
2409.13
2157.98 2305.99
2271.03 2246.05 2335.07
2000
0
1000
1938.82
2689.16 2782.32
2549.08
3072.44
2717.15
2458.04
2569.17
2944.36
2799.29
3237.47
3312.34
2984.75
3312.38
3072.42 3174.52 3278.52
3223.35
3092.34
2062.93
1993.88
1374.51
1124.57
1819.90
2125.97
785.33
1439.73
40000
1029.59
1274.60
55000
1916.83
1594.70
1226.58
60000
2500
3000
3500
Mass
3500
Mass
C l u s te r A r e a
40000
35000
1904.94
30000
MS - INTERNAL CALIBRATION
Plate: 1 Spot: H7
25000
170000
160000
842.51
20000
927.50
150000
100
745.30
15000
1479.78
1193.62
1107.55
10000
760.37
130000
962.53 1062.54
1003.59
786.30
896.46
1234.67
1161.62 1320.60
1493.74
1707.78
1346.64
1179.62
1996.91
1418.67
1747.78
1277.69
1043.54
90
1532.80
1387.71
1564.75
1612.81
1771.87
1678.84
1791.73
2139.00
2001.92
2334.98
3085.39
2545.17
2741.48
3035.25
3247.40
3246.52
3576.11
3959.72
0
1088.51
1000
1500
2000
2500
3000
3500
Mass
1003.58
100000
C l u s te r A r e a
2211.10
1858.91
1592.83
1298.63
110000
220000
1366.61
1.5E+4
1439.77
1082.59
5000
120000
MS - INTERNAL CALIBRATION
200000
1298.651( R 11281,S906)
1920.91
1639.92
816.45
140000
80
90000
1210.59
80000
180000
870.54
1179.58
70000
898.44
70
842.51
160000
60000
830.45
976.56
MS - INTERNAL CALIBRATION
50000
1475.73
927.49
140000
1029.57
1905.83
1287.63
1136.60
40000
280000
1791.71
1427.66
C l u s te r A r e a
1612.76
1213.63
120000
1088.52
1003.59
1377.59
797.38
30000
1993.86
2125.97
2383.97
1770.90
1914.85
778.44
2062.92
896.47
1060.55
20000
1275.64
770.36
240000
1337.67
1191.52
1639.91
1505.69
1707.76
2211.11
1835.94
1584.71
977.49
1657.77
1392.58
10000
2496.36
1900.82
1456.66
1261.55
1107.54
100000
1740.79
1676.71
1947.89
1985.92
2093.96
2400.05
2289.99
2226.13 2329.08
2541.20
2612.21
2478.06 2575.25
1000
1500
2000
2500
1901.838( R 17349,S332)
2123.04
870.54
880.417( R 8953,S490)
1179.61
927.49
719.36
180000
1195.57
898.44
1298.65
40
1458.70
1402.65 1493.74
1144.57
1034.59
1070.61
745.29
40000
160000
1003.58
842.51
1906.829( R 16997,S261)
1320.59
1707.78
1765.75
1586.79
1088.52
2281.04
1639.94
2652.20
1835.95
140000
1377.62
1118.55
1880.93
1434.76 1529.68
842.51
1559.77
120000
1684.88
1646.75
1136.61
1791.80
1811.95
1740.72
2383.96
1951.97
2211.11
1993.88
2717.10 2815.39
2343.00
2438.27
2264.59
2035.00
2060.94
880.42
2215.12
1918.85 2006.01
1003.58
65000
898.442( R 7544,S370)
1612.82
940.49
808.40
20000
70000
1088.519( R 9327,S508)
1210.607( R 10791,S460)
3500
1235.69
60000
75000
50
3223.43
3237.64
3000
Mass
200000
MS - INTERNAL CALIBRATION
1298.64
3073.51
2717.14
80000
1210.60
80000
3312.45
2782.36
2705.22
1856.84
1531.68
220000
85000
60
719.37
1298.65
260000
C l u s te r A r e a
0
699.0
1237.640( R 9786,S113)
2497.343( R 19048,S128)
1820.900( R 16302,S79)
1029.588( R 7897,S79)
1393.604( R 10852,S60)
2291.005( R 18508,S64)
1794.814( R 17757,S38)
921.478( R 8410,S55) 1412.212( R 6599,S20)
2690.148( R 17772,S47)
1997.895( R 14319,S29)
3074.423( R 15546,S48)
807.399( R 7419,S23)
1537.693( R 7392,S11)
2673.131( R 17546,S15)
1949.876( R 10399,S12)
3481.632( R 16563,S16)
2565.20
2461.19
2612.17
2579.22
1000
100000
1500
2000
2500
2975.43
3000
3132.57
1377.615( R 11118,S185)
3500
Mass
1029.58
1771.929( R 16092,S138)
1905.83
921.48
1493.73
1427.68
1140.54
1377.61
796.36
50000
1770.92
1237.64
60000
1179.59
1639.93
816.49
1639.94
1329.64
1657.79
1359.61
1537.77
1000
1500
785.34
1107.54
1993.87
1791.73
2125.97
3237.45
1456.68
3000
3500
1880.92
2496.32
2062.92
1521.68
1740.71
1684.84
1820.900( R 16302,S79)
2497.343( R 19048,S128)
1361.8
2024.6
2687.4
Mass (m/z )
1770.91
1594.68
1419.67
1985.98
2290.01
1815.88
2717.10
2282.04
2211.10
1854.81
3086.43
2248.02
1561.74
2542.21 2646.13
2084.00
2567.13
2780.27
2686.20
3073.36
3011.60
3312.30
3353.17
0
1500
2500
1029.588( R 7897,S79)
1393.604( R 10852,S60)
2291.005( R 18508,S64)
1794.814( R 17757,S38)
921.478( R 8410,S55) 1412.212( R 6599,S20)
2690.148( R 17772,S47)
1997.895( R 14319,S29)
3074.423( R 15546,S48)
807.399( R 7419,S23)
1537.693( R 7392,S11)
2673.131( R 17546,S15)
1949.876( R 10399,S12)
3481.632( R 16563,S16)
2383.96
1707.78
1357.71
1065.55
5000
1000
3072.47
2817.24
0
699.0
1905.83
20000
1254.61
2717.09
2782.30
2688.21
Mass
1427.68
980.50
2383.96
2211.10
2289.99
2078.91
2172.10 2282.08
1880.92
1923.99 2034.97
2247.95
1854.85
2000
1612.77
1029.58
761.41
1707.78
1584.72 1677.80
30000
10000
10
1819.89
1392.57
1071.50
830.45
1377.61
1791.73
1479.80
1264.70
982.45
20000
948.43
15000
1237.640( R 9786,S113)
1456.68
719.37
745.29
880.42
25000
1136.615( R 9246,S155)
2125.97
2062.93
1191.53
40000
1149.57 1320.59
40000
1234.67
2496.33
1505.71
1210.60
1088.52
898.44
1993.87
1612.77
745.32
45000
35000
20
80000
55000
1994.885( R 18017,S233)
2126.984( R 17889,S218)
30
2778.50
2681.31
2495.27
2364.23
2139.04 2297.03
927.49
60000
C l u s te r A r e a
10
MS - DEFAULT CALIBRATION
2000
2500
Mass
3000
3500
3350.2
4013.0
Bioinformatic analysis
Swiss-Prot
(Reviewed)
Manual anotation
trEMBL
(Unreviewed)
Automatic anotation
Plate: 1 Spot: G8
MS spectrum
150000
842.51
1337 Da
140000
130000
1306.64
1156.59
120000
1337.69
110000
100000
1141.59
C l u s te r A r e a
90000
80000
70000
2211.11
60000
1268.69
999.53
1360.58
50000
1582.81
1430.75
787.51
810.53
2005.99
1280.69
846.49
40000
1058.52
1470.70
1201.63
742.47
1701.88
1373.67
1410.72
30000
1238.60
1045.56
1833.95
880.43
1635.80
896.43
1676.83
20000
1169.55
912.43
759.46
10000
2282.02
1791.86
1775.86
1453.68
1496.80
1110.54
1554.78
1007.46
950.48
1094.55
1212.57
1649.80 1728.85
2153.91
1874.02
1818.96
1940.91
1886.90
1613.72
1525.71
2390.21
2122.11
1744.84
973.49
1570.82
1920.95
2032.96
1947.99
2366.28
2225.11
2164.96
2039.98
2299.17
2192.13
2315.14
2255.15
1992.94 2072.02
2407.14
2349.26
2455.35
2587.33
2650.30
2802.66
2952.53
2796.43
0
800
1000
1200
1400
1600
1800
2000
Mass
2200
2400
2600
2800
3000
3200
1337 Da
Collision cell
Intensity
m/z (Da)
Plate: 1 Spot: G8
70000
Peptide sequencing
F, a1
65000
60000
MS/MS spectrum
55000
50000
L
1337 Da
y1
45000
C l u s te r A r e a
1337.51
40000
a2
35000
30000
25000
20000
112.03
y4, VSTPL(b)
NQ(b)
15000
b2
PL(b)
NQV(b)
229.02
b3, TPLL(b)
TP(b), LL(a)
10000
442.08
171.03
496.18
155.02
y2 - NH3
5000
141.04
y4 - NH3
185.04
b4
839.34
YNQ(b)
y5, NQVSTP(a)
297.03
b5
1293.39
1329.75
967.41
y6
STPL(b)
914.30
0
100
200
300
400
500
600
700
Mass
800
900
1000
1100
1200
1300
MS/MS spectrum
http://www.expasy.org/proteomics
http://prospector.ucsf.edu/prospector/mshome.htm
www.uniprot.org
NCBI
www.ncbi.nlm.nih.gov
www.matrixscience.com
PeptideMass
http://web.expasy.org/peptide_mass/
PeptideCutter
http://web.expasy.org/peptide_cutter/